The basis for many molecular-biological and immuno assays, diagnostic assays and tests, among other things, include the steps of obtaining a cellular sample (e.g., blood, tissue, etc.), separating out the cellular material of interest, disrupting or lysing the cells of interest to release the crude DNA and RNA (for simplicity, a reference to DNA in the following text also refers to RNA where appropriate) all protein, purifying the crude lysate (i.e. removing cellular debris), and performing some enzymatic reaction to analyze the lysate as desired.
Dielectrophoresis has become a popular technique for separating microparticles which are either charged or uncharged in solution. Techniques reported prior to this invention are almost always performed in a glass slide based device having exposed (i.e. naked) interdigitated electrodes plated on the surface of the slide and having a flow chamber with a volume of several hundred microliters. Cells are separated in such devices based on their dielectric properties by choosing separation buffer(s) with appropriate conductivity and an AC signal with a suitable amplitude and frequency. These prior devices have several problems, including the following. A first problem is that both separated and unseparated cells bind nonspecifically to the exposed glass surface of the slide and to the exposed electrode surfaces. A second problem is that the volume of the flow chamber (several hundred microliters) is so large that thermal convection disturbs and pushes off cells initially retained by the electrodes. A third problem is that washing off any undesired cells is not easily accomplished without disturbing the cells that are desirably retained on the electrodes, as the desired cells and electrodes stand in the way of fluidic flow and, hence, block the wash flow containing any undesired cells.
Disrupting or lysing cells releases the crude DNA and RNA material along with other cellular constituents. Electronic cell lysing techniques reported prior to this invention are conventionally performed by applying a series of high voltage DC pulses in a macrodevice, as opposed to a microchip-based device. These conventional electronic lysis techniques have several problems, including the following. A first problem is that the electronic lysis conditions specified by commercial macro-device do not release DNA molecules of high molecular weight (larger than 20 Kb) because the high molecular weight DNA molecules do not fit through the pores created in the cell membrane by the prior lysing methods. A second problem is that some nucleic acids originally released in the lysis chamber are lost due to their non-specific binding to the surface of the lysis chamber. A third problem is that the conventional electronic lysis macrodevice works as a stand alone unit such that both dielectrophoretic cell separation and electronic lysis cannot be performed on the same module.
The crude lysate is then purified (i.e., undesired cellular debris is washed off or separated), and then the purified lysate is subjected to enzymatic reaction(s) to prepare the lysate for hybridization, detection, and analysis. Such reactions may include, for example, denaturing, cleaving, or amplifying the lysate. Only after these sample preparation and DNA processing steps, the actual hybridization reaction is performed, and, finally, detection and data reduction convert the hybridization event into an analytical result. These conventional preparation and processing techniques have several problems, including the following. A first problem is that the steps of sample preparation and processing are typically performed separately and apart from the other main steps of hybridization, detection and analysis. In addition, most of these techniques involve carrying out numerous operations (e.g., pipetting, centrifugations, electrophoresis) on a large number of samples. They are often complex and time consuming, and generally require a high degree of skill. Many a technique is limited in its application by a lack of sensitivity, specificity, or reproducibility. For example, these problems have limited many diagnostic applications of nucleic acid hybridization analysis.
Attempts have been made to use dielectrophoresis to separate and identify cells. For example, U.S. Pat. No. 4,326,934 to Herbert discloses a method and apparatus for cell classification by continuous dielectrophoresis. Cells were separated by making use of both the positive and negative dielectrophoretic movement of cell particles. Separated cells were allowed to be characterized and/or classified by viewing the characteristic deflection distance of cells moving through the two electrodes.
Also, U.S. Pat. No. 5,344,535 to Walter et al. discloses a method and apparatus for the characterization of micro-organisms and other particles by dielectrophoresis. Cells were characterized by matching their signature dielectrophoretic collection rates.
And U.S. Pat. No. 5,569,367 to Walter et al. discloses a method and apparatus for separating a mixture using a pair of interdigitated electrodes. The apparatus used two energized interdigitated electrodes that obstruct straight through flow of cells and further separate different types of cells into fractions by applying a non-uniform alternating field. The electrode structure is comprised of interleaved grid-like structures aligned to obstruct flow through the structure.
As exemplified above, several methods of separating cells by dielectrophoresis have been developed. In dielectrophoresis migration, dielectrophoretic forces are applied to drive the migration of different types of cells within the biological sample to different regions of the microelectrode structure. In dielectrophoresis retention, competition between dielectrophoretic forces and fluid-flow forces is exploited to selectively trap cells of interest from a biological sample at electrodes and hold those cells against an imposed fluid-flow stream. Although dielectrophoresis retention has been successfully demonstrated for isolating cancer cells, bacteria, or CD34+ stem cells from blood, two practical deficiencies minimize the effectiveness of this technique in biomedical applications. First, conventional dielectrophoretic systems often consist of a thin, low volume flow chamber utilizing a two-dimensional array of microelectrodes disposed at the bottom of the flow chamber. However, with increasing distance (e.g., more than 10 μm) from the electrode plan, the electric field decays rapidly and the non-uniformity of the field distribution becomes smaller. The dielectrophoretic forces in such two-dimensional systems are only able to collect the cells of interest that are located close to the electrode plane, while cells floating well above the electrode plane can be lost during washing. Second, the dielectrophoretic forces acting on the cells are not the uniform due to the heterogeneous properties among different cells types. Therefore, cells that experience smaller dielectrophoretic properties may be lost during washing steps because the dielectrophoretic forces are not strong enough to hold the cells against the fluid-flow forces.
In addition, attempts have been made to combine certain processing steps or substeps together. For example, various microrobotic systems have been proposed for preparing arrays of DNA probes on a support material. For example, Beattie et al., in The 1992 San Diego Conference: Genetic Recognition, November, 1992, used a microrobotic system to deposit micro-droplets containing specific DNA sequences into individual microfabricated sample wells on a glass substrate. Various attempts have been made to describe integrated systems formed on a single chip or substrate, wherein multiple steps of an overall sample preparation and diagnostic system would be included. For example, A. Manz et al., in “Miniaturized Total Chemical Analysis System: A Novel Concept For Chemical Sensing”, Sensors And Actuators, B1(1990), pp. 244-248, describe a ‘total chemical analysis system’ (TAS) which comprises a modular construction of a miniaturized TAS. Sampling, sample transport, any necessary chemical reactions, chromatographic separations as well as detection were to be automatically carried out. Yet another proposed integrated system is Stapleton, U.S. Pat. No. 5,451,500, which describes a system for automated detection of target nucleic acid sequences in which multiple biological samples are individually incorporated into matrices containing carriers in a two-dimensional format. Different types of carriers are described for different kinds of diagnostic tests or test panels.
Various multiple electrode systems are disclosed which purport to perform multiple aspects of biological sample preparation or analysis. Pace, U.S. Pat. No. 4,908,112, entitled “Silicon Semiconductor Wafer for Analyzing Micronic Biological Samples” describes an analytical separation device in which a capillary-sized conduit is formed by a channel in a semiconductor device, wherein electrodes are positioned in the channel to activate motion of liquids through the conduit. Pace states that the dimension transverse to the conduit is less than 100 μm. Pace also states that all functions of an analytical instrument may be integrated within a single silicon wafer: sample injection, reagent introduction, purification, detection, signal conditioning circuitry, logic and on-board intelligence. Soane et al., in U.S. Pat. No. 5,126,022, entitled “Method and Device for Moving Molecules by the Application of a Plurality of Electrical Fields”, describes a system by which materials are moved through trenches by application of electric potentials to electrodes in which selected components may be guided to various trenches filled with antigen-antibodies reactive with given charged particles being moved in the medium or moved into contact with complementary components, dyes, fluorescent tags, radiolabels, enzyme-specific tags or other types of chemicals for any number of purposes such as various transformations which are either physical or chemical in nature. It is said that bacterial or mammalian cells; or viruses may be sorted by complicated trench networks by application of potentials to electrodes where movement through the trench network of the cells or viruses by application of the fields is based upon the size, charge or shape of the particular material being moved. Clark, U.S. Pat. No. 5,194,133, entitled “Sensor Devices”, discloses a sensor device for the analysis of a sample fluid which includes a substrate in a surface of which is formed an elongate micro-machined channel containing a material, such as starch, agarose, alginate, carrageenan or polyacrylamide polymer gel, for causing separation of the sample fluid as the fluid passes along the channel. The biological material may comprise, for example, a binding protein, an antibody, a lectin, an enzyme, a sequence of enzymes, or a lipid.
Various devices for eluting DNA from various surfaces are known. Shukla U.S. Pat. No. 5,340,449, entitled “Apparatus for Electroelution” describes a system and method for the elution of macromolecules such as proteins, DNA and RNA from solid phase matrix materials such as polyacrylamide, agarose and membranes such as PVDF in an electric field. Materials are eluted from the solid phase into a volume defined in part by molecular weight cut-off membranes. Okano, U.S. Pat. No. 5,434,049, entitled “Separation of Polynucleotides Using Supports Having a Plurality of Electrode-Containing Cells” discloses a method for detecting a plurality of target polynucleotides in a sample, the method including the step of applying a potential to individual chambers so as to serve as electrodes to elute captured target polynucleotides, the eluted material is then available for collection.
Generally, the prior art processes have been extremely labor and time intensive. Multiple steps requiring human intervention either during the process or between processes are suboptimal in that there is a possibility of contamination and operator error. Further, the use of multiple machines or complicated robotic systems for performing the individual processes is often prohibitive except for the largest laboratories, both in terms of the expense and physical space requirements.
As is apparent from the preceding discussion, numerous attempts have been made to provide effective techniques to conduct sample separation and preparation reactions. However, for the reasons stated above, these techniques are limited and lacking. These various approaches are not easily combined to form a system which can carry out a complete DNA diagnostic assay. Despite the long-recognized need for such a system, no satisfactory solution has been proposed previously.
There is a continuing need for methods and devices which lead to improved dielectrophoretic separation of biological cells as well as improved biological stability of the separated cells. There is also a continuing need for methods and devices which improve cell preparation and analysis, and which are capable of integrating cell separation, preparation, and analysis in a single system.